Non-enzymatic, salt-mediated synthesis of polynucleic acids

ABSTRACT

Provided herein is a method for synthesizing polynucleic acids, comprising the steps of (a) providing an acidic solution substantially free of nucleic acid polymerase and lipids, but containing mononucleotides and a monovalent salt; (b) drying and resolubilizing the mixture of step (a) a plurality of times; and (c) recovering polynucleic acids from a resolubilized mixture of step (b). In certain aspects, the method further uses a low pH, e.g. about 3; it can utilize monophosphates, such as AMP rather than ATP; and it can be used with a polynucleotide template to form a sequence at least partially complementary to said template. Thus, both single-stranded and double-stranded polynucleic acids are provided. Ammonia salts have been used to obtain RNA lengths from 10 to 300 nucleotides after 16 half hour cycles and an effective temperature includes between 80° C. and 100° C.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/022,487 filed on Mar. 16, 2016, now U.S. Pat. No. 10,280,191, whichapplication is a national phase filing of PCT Patent Application No.PCT/US2014/055951 filed on Sep. 16, 2014, which application claimspriority to U.S. Provisional Patent Application No. 61/879,046, filed onSep. 17, 2013, which is hereby incorporated by reference in itsentirety.

STATEMENT OF GOVERNMENTAL SUPPORT

None.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT

In accordance with “Legal Framework for EFS-Web,” (6 Apr. 11) Applicantssubmit herewith a sequence listing as an ASCII text file. The text filewill serve as both the paper copy required by 37 CFR 1.821(c) and thecomputer readable form (CRF) required by 37 CFR 1.821(e). The date ofcreation of the file was Sep. 16, 2014, and the size of the ASCII textfile is 1008 bytes. Applicants incorporate the contents of the sequencelisting by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of chemical (abiotic)synthesis of polynucleic acids, particularly single stranded and doublestranded nucleic acids, such as double stranded RNA.

RELATED ART

Presented below is background information on certain aspects of thepresent invention as they may relate to technical features referred toin the detailed description, but not necessarily described in detail.That is, individual compositions or methods used in the presentinvention may be described in greater detail in the publications andpatents discussed below, which may provide further guidance to thoseskilled in the art for making or using certain aspects of the presentinvention as claimed. The discussion below should not be construed as anadmission as to the relevance or the prior art effect of the patents orpublications described.

It has been reported that RNA-like molecules are synthesized fromordinary mononucleotides if the monomers are organized within a liquidcrystalline matrix (Rajamani S, Vlassov A, Benner S, Coombs A,Olasagasti F, Deamer D., “Lipid-assisted synthesis of RNA-like polymersfrom mononucleotides,” Orig Life Evol Biosph. 38:57-74 (2008)). Thismethod is based on providing an aqueous suspension of phospholipidvesicles and monomers, and subjecting the mixture to alternating cyclesof hydration and dehydration, hereafter referred to as HD cycles.

It has also been demonstrated that under the same conditions sequenceinformation could be transferred non-enzymatically from a templatestrand of DNA to product strands (Olasagasti F, Kim H J, Pourmand N,Deamer D W. 2011, “Non-enzymatic transfer of sequence information underplausible prebiotic conditions,” Biochimie. 93:556-61). In this report,an environment was created where dry and wet periods were cycled. Underanhydrous conditions, lipid molecules present in the medium could formfluid lamellar matrices and work as organizing agents for thecondensation of nucleic acid monomers into polymers on a DNA templatestrand.

Toppozini L, Dies H, Deamer D W, Rheinstädter M C (2013) AdenosineMonophosphate Forms Ordered Arrays in Multilamellar Lipid Matrices:Insights into Assembly of Nucleic Acid for Primitive Life. PLoS ONE 8:e62810 discloses the use of X-ray diffraction analysis to show thatadenosine monophosphate forms a two-dimensional ordered array when itundergoes the dehydration step of an HD cycle within a multilamellarmatrix of dimyristoyl-phosphatidylcholine.

Deamer U.S. Pat. No. 7,772,390, entitled “Lipid mediated nucleic acidsynthesis,” issued Aug. 10, 2013, discloses methods in which lipids andmonomeric precursors, e.g., mononucleotides, of the desired polymericproducts are combined to produce a reaction mixture. The reactionmixture is then subjected to one or more steps of drying and rehydratingto produce a desired polymeric product, e.g., nucleic acid.

Rajamani et al. U.S. Pat. No. 8,314,209, entitled “Lipid-assistedsynthesis of polymer compounds and methods for their use,” issued Nov.20, 2012, discloses a method that provides for the synthesis ofpolynucleotides from mononucleotides in the absence of catalyticenzymes. The method comprises providing an aqueous solution having aplurality of phospholipid molecules and monomer molecules; subjectingthe aqueous suspension to fluctuating cycles of drying and hydratingconditions at elevated temperature ranges; subjecting the aqueoussolution to fluctuating [H+] conditions; the fluctuating conditionsthereby allowing formation of a chemical bond between at least twomonomers to create a polymer.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The present invention pertains to a method for in vitro method forsynthesizing polynucleic acids (“polynucleotides”) from mononucleotideprecursors using monovalent salts as organizing agents and otherconditions. According to the present invention, double stranded andsingle RNA may be produced. The RNA produced by the present methods hasbeen shown to be polymeric and of a native structure sufficient to bedigestible by RNase A.

In addition, the present methods may be used for replication of apolymeric nucleotide template, using a non-enzymatic polymer chainreaction.

The present invention comprises an in vitro method for synthesis andrecovery of polynucleotides, wherein said polynucleotides have adirected sequence, based on a template polynucleotide added to thereaction solution. In addition, the invention contemplates the creationof libraries of polynucleotides, such as RNA polymers, wherein thelibrary contains a diverse population of different sequences. Suchlibraries are useful, for example, in the identification of RNA or DNAaptamers, and the study of RNA diversity in evolution.

Thus, the present invention may comprise, in certain aspects, a methodfor synthesizing polynucleic acids, comprising: providing an acidicsolution substantially free of polymerase and lipids, but containingmononucleotides and a dissolved monovalent salt in high concentration;drying and resolubilizing the solution a plurality of times; andrecovering polynucleic acids from a resolubilized mixture.

The present invention may comprise, in certain aspects, a method forsynthesizing RNA polynucleic acids, comprising the steps of: (a)providing an acidic solution containing 5′-mononucleotides such asadenosine monophosphate (A), uridine monophosphate (U), guanosinemonophosphate (G) and cytidine monophosphate (C) in acidic form,together with a monovalent salt, such as a metal-halogen salt in highconcentration; (b) drying and resolubilizing the solution of step (a) aplurality of times; and (c) recovering polynucleic acids from aresolubilized mixture of step (b). The nucleotides used do not requirechemical activation and may contain a native or natural structure at the5′ monophosphate terminus.

The present invention may comprise, in certain aspects, a method asreferred to above wherein the acidic solution is adjusted to a pHbetween 2 and 4 during the synthesis of the polynucleic acid accordingto the present process. The present invention may comprise, in certainaspects, a method as referred to above wherein the mononucleotides addedto the mixture are monophosphates. The present invention may comprise,in certain aspects, a method as referred to above wherein themononucleotides comprise A, U, G, and C in various combinations. Themononucleotides added to the solution may be added in a ratio of equalamounts of each of the four bases, or certain bases may be omitted orincreased, depending on the goals of the synthesis. In cases of DNAsynthesis, deoxyribonucleotides and thymidylic acid (T) may also be usedin place of U. The present invention may comprise, in certain aspects, amethod as referred to above comprising the step of adding apolynucleotide template to the solution and said wherein recoveringcomprises recovering polynucleic acids produced in the process having asequence at least partially complementary to said template.

The salt or salts used in the present method may be present in a rangeof concentrations, up to saturation. In certain embodiments, themonovalent salt(s) are present in a concentration in solution of from0.05 and 2M or from 0.001 to 3M. or from 0.05 to 1M (See “Ranges” forfurther variations).

The present invention may also comprise, in certain aspects, a method asreferred to above wherein the step of recovering polynucleic acidsthereby produced comprises recovering double stranded polynucleic acids,that is, partially or fully double-stranded, e.g. as shown in FIG. 12.The present invention may comprise, in certain aspects, a method asreferred to above wherein the double stranded polynucleic acids arepredesigned according to a certain sequence and be a short interferingRNA.

The above-mentioned recovering may comprise recovering polynucleotideshaving a sequence length greater than 20 bases, greater than 100 bases,or greater than 200 bases

The present invention may also comprise, in certain aspects, a method asreferred to above wherein the monovalent salt is selected from the groupconsisting of NaF, CsCl, NaBr, NaClO₄, NaCl, KCl, and NH₄Cl. The presentinvention may comprise, in certain aspects, a method as referred toabove wherein the salt is an ammonium salt (e.g. NH⁴⁻halo), atetramethylammonium salt or another halide salt (including andadditional to the halide salts listed above).

The present invention may also comprise, in certain aspects, a method asreferred to above further comprising the step of heating the solution,e.g. to a temperature between 80° C. and 100° C.

The above methods are abiotic, that is, without use of biologicalmechanisms such as cells or polymerase. The above methods are preferablycarried out under anaerobic conditions. The present methods are alsoindependent of the use of organizing macromolecules such as lipids orsimilar lamellar matrices. In certain embodiments, the present inventioncomprises the use of an automatable device that holds the reactants fora defined period, controlling heating (drying) and resolubilization withsalt solutions, and further controlling the atmosphere in the device tobe low in oxygen, e.g. creating a CO₂ atmosphere.

In certain embodiments, the present invention comprises an in vitromethod for synthesizing polynucleic acids from a plurality ofmononucleotides, comprising the use of an acidic solution substantiallyfree of polymerase and lipids, but containing mononucleotides and amonovalent salt at a concentration of at least 0.01 M; drying thesolution; resolubilizing the solution; and repeating drying andresolubilizing steps a plurality of times. Afterwards, the result willbe the formation of bonds between the mononucleotide. Then, the methodcomprises recovering (i.e. separating) polynucleic acids from aresolubilized mixture.

In certain embodiments, the present invention comprises heating thesolution during drying. In certain embodiments, the present inventioncomprises the use of an acidic solution adjusted to a pH between 2 and4, or between 2 and 6. In certain embodiments, the present inventioncomprises the addition to the solution of mononucleotides aremonophosphates (as opposed e.g. to salts) and the mononucleotides arepresent at a concentration of at least 1 millimolar. In certainembodiments, the present invention comprises the use of mononucleotidescomprising A, U or T, G, and C, using the standard abbreviations for DNAand RNA bases.

In certain embodiments, the present invention comprises the use of apolynucleotide template. The present methods then will also compriserecovering polynucleic acids having a sequence at least partiallycomplementary to said template. The step of recovering polynucleic acidsmay comprise recovering double stranded polynucleic acids, which may bepartially or completely double stranded.

In certain embodiments, the present invention comprises the in vitrosynthesis of double stranded polynucleic acids are short interferingRNAs.

In certain embodiments, the present invention comprises the use of amonovalent salt is one or more selected from the group consisting ofNaF, CsCl, NaBr, NaClO4, NaCl, KCl, and NH4Cl and said saltconcentration is between 0.05 M and 2M in solution. In certainembodiments, the salt is a tetramethylammonium salt. In certainembodiments the salt is a halide (halogen salt).

In certain embodiments, the present invention comprises the step ofheating the solution. In certain embodiments the heating is to atemperature between 80° C. and 90° C.

In certain embodiments, the present invention comprises methods asdescribed above wherein the recovering comprises recoveringpolynucleotides having a sequence length greater than 200 bases.

In certain embodiments, the present invention comprises methods asdescribed above wherein the reaction is carried out under anaerobicconditions.

In certain embodiments, the present invention comprises methods asdescribed above wherein the monovalent salt concentration is between0.05 and 2M in solution.

In certain embodiments, the above-described methods result in inventivesystems. That is, the invention may comprise or consist essentially of asystem for use in preparing a polynucleotide of a desired sequence,comprising: an solution adjusted to be between pH 2 and 4, free ofpolymerase and lipids; said solution comprising a mononucleotides A, U,G, and C, each at a concentration of from 0.001 to 3M; a monovalent saltselected from the group consisting of NaF, CsCl, NaBr, NaClO4, NaCl,KCl, and NH4Cl; and a template polynucleotide complementary to thedesired sequence; said solution further maintained in the system at ananerobic environment. The invention in certain aspects comprises asystem for use in preparing polynucleotides, comprising or consistingessentially of: a dried solution, free of polymerase and lipids; saiddried solution comprising a mononucleotides A, U, G, and C, each at aconcentration of from 0.001 to 3M if the liquid; a monovalent saltselected from the group consisting of NaF, CsCl, NaBr, NaClO4, NaCl,KCl, and NH4Cl; and a template polynucleotide complementary to thedesired sequence; said dried solution further maintained in the systemat an anerobic environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, 1B, 1C: FIG. 1A is a schematic diagram showing formation ofphosphodiester bonds between monomers in acid form (rather than saltform) in the presence of a monovalent salt as used in the presentmethods. As can be seen, an H+ will react with the P═O, then a waterleaves when the phosphodiester bond is formed. FIG. 1B illustrateshydrogen bonding between a mononucleotide illustrated in FIG. 1A and auracil monophosphate; FIG. 1C shows the arrangement of nucleotideswithin a semi-crystalline matrix established by the salt concentrationin the present methods. The schematic indicates the edge of a saltcrystal. The reaction takes place in the concentrated salt solutionbetween the crystals. Bases 1, 2 and 3 may be added in oligomeric formas template upon which a second strand is formed in a complementarysequence.

FIG. 2 is a photograph of a chamber that cycles potential reactantsbetween hydrated and dehydrated states. There are 24 wells in thecentral disk that hold reactants, and a stepper motor rotates the disk15 degrees every 30 minutes. The rotation brings the samples over twoheat sources, and dry carbon dioxide flows into four sample wells oneach side of the disk to aid dehydration. The chamber is filled withcarbon dioxide to exclude oxygen, and water is delivered to samples by aprogrammable syringe pump.

FIG. 3A, 3B, 3C is a series of photographs of a small HD (heat and dry)apparatus in which cycling is carried out by hand. The samples arecontained in two wells in each slide, so that 8 samples can be processedin parallel. To aid drying, carbon dioxide gas is delivered into thewells through the flow box shown on the right.

FIG. 4A, 4B, 4C is a photograph (4A) scan (4B) and graph (4C) showinghydrolysis of poly AU. The gels and scans are duplicate samples exposedto four HD cycles. The experiment began with 20 micrograms of polyAU,and the Y axis shows the micrograms of polymer remaining after eachcycle.

FIG. 5 is a photograph of a gel showing polymerization of AMP and UMP.It demonstrates the effect of cycles of HD on yield. A fluorescent dye(ethidium bromide) is used to stain the products of the reaction. Theimage is inverted so that the fluorescence is shown as dark against alight background.

FIG. 6 is a photograph of a gel showing polymerization in the presenceof different salts.

FIG. 7 is a photograph of a gel showing the effect of pH on the productsof 4 HD cycles run with AMP and UMP as monomers. A low pH is requiredfor the reaction to proceed.

FIG. 8 is a diagram showing acid-catalyzed bond formation proposed as apolymerization mechanism in the present process.

FIG. 9 is a photograph of a gel showing that RNase A cleaves thepresently produced polymers, with longer strands more sensitive to theenzyme's action.

FIG. 10 is an image from atomic force microscopy showing evidence ofduplex RNA.

FIG. 11A, 11B shows a gel (11A) and scan (11B) of longer polymers(indicated by box in 11A) showing the products of a feeding experiment.

FIG. 12 is a diagram showing possible dsRNA products synthesized by HDcycles. The lengths and sequences shown are arbitrary, and are forillustration purposes only.

FIG. 13A, 13B is a pair of micrographs showing salt crystals formedunder conditions of the present process, showing the possibility thatthe salts in the process organize the nucleotides. These experimentsused 50 μl 0.1M NH₄Cl+5 mM AMP+5 mMUMP+0.1 mM pyramine, dried one hourat 85 deg. C. FIG. 13A is a phase image; FIG. 13B is a fluorescentimage, excited by 360 nM UV illumination. 160× original magnification.

FIG. 14 is a histogram showing yields obtained from different salts.Lane A: NH₄Cl; lane B: C₁₉H₄₂BrN; lane C: HCO₂NH₄; lane D:(NH₄)₆Mo₇O₂₄4H₂O; lane E: NH₄H₂PO₄.

FIG. 15 is a line graph RNA kinetics of oligomerization experiments.Mixture of AMP 10 mM+UMP 10 mM+NH4Cl 0.1 M (1:1:1 volume ratio) showingthe total amount and yield of products over multiple cycles. Each hourhas two 30 minutes cycles, so 40 hours represents 80 cycles.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Overview

The present invention pertains to the non-enzymatic synthesis ofpolynucleic acids from short precursor molecules, preferably nativenucleic acid monomers. The present methods are carried out in vitro,i.e. without the use of cells or other organisms; they are carried outunder synthetic conditions. They are applicable to formingsingle-stranded and double-stranded RNA and DNA from pre-definedmixtures of mononucleotides under defined conditions described below. Animportant feature of the present method is the ability of the method toproduce multiple copies of polynucleotides of defined sequence based ona template sequence. That is, multiple copies of a polynucleotide (DNAor RNA) having a predefined sequence may be made. The template strands(which may be the same or different sequences) are added along with thenecessary monomers (A, U, G, C, or, in the case of DNA, T). Also addedare monovalent salts and a pH lowering agent (acid). The monomers areadded in their free acid monophosphate form, e.g. adenosinemonophosphate, uracil monophosphate, guanine monophosphate, thyminemonophosphate, etc. No enzymes or lipids are used for thepolymerization. Accordingly, non-natural bases may also be incorporatedinto the template and/or the resulting copies.

A representative reaction of 5′ uridine monophosphate 102 with a 5′phosphate group 103, i.e. in acidic form, as also shown at 104 and 5′adenosine monophosphate 106 is shown in FIG. 1A. The reaction occurs inan aqueous environment with a monovalent salt, illustrated as NaCl at108, but which could also be ammonium chloride, etc. Nucleotides 102 and106 combine to form a phosphodiester bond as indicated by arrow 109,forming a 3′-5′ linkage. A third 5′ monophosphate base 110 can also forma 3′-5′ linkage with the base 106, and so forth with other monomers.This forms a single stranded polynucleotide. In certain instances, a2′-5′ linkage may be formed. Evidence for this polynucleotide structureis provided by RNase A experiments. RNase A cleaves specifically afterpyrimidine nucleotides. Cleavage takes place in two steps: first, the3′, 5′-phosphodiester bond is cleaved to generate a 2′, 3′-cyclicphosphodiester intermediate; second, the cyclic phosphodiester ishydrolyzed to a 3′-monophosphate.

As shown in FIG. 1B, the polymerization scheme shown in FIG. 1A can beused to form a template molecule 202 to which complementary bases 204can be formed, with base pairing as shown by dotted lines 206. Asdescribed below, preformed templates can be added to direct synthesis toform a desired sequence.

As shown in FIG. 1C, the salt concentration in the mixture of thepresent method is thought to drive the monophosphate nucleotides intoclose proximity, and also into an orientation where base pairing andbase stacking can occur. In this orientation, the phosphate groups arein close proximity to the —OH groups of a neighboring ribose, anarrangement conducive to synthesis of phosphodiester bonds that link theAMP and UMP monomers into a nucleic acid polymer. Also shown is theformation of dsRNA; in certain embodiments, described below, bases 1, 2and 3 will be added by pairing with a template strand rather than asindividual monomers. This is indicated by a dashed line betweenphosphate and hydroxyl groups in bases 1, 2 and 3. A phosphate linkagewill also form between the third carbon of one ribose and the fifthcarbon of the next ribose in pairing with the template.

The present methods can be used to produce oligonucleotides of 300 nt ormore. The present methods are characterized in that they do not employenzymes such as are conventionally used in synthesizingoligonucleotides, and they do not employ lipids to concentrate thenucleotides. To reiterate, no enzymes (e.g. polymerase) or otherbiological structures are added to the reaction mixture. Thus, thereaction solution may be free of enzyme co-factors. The term co-factorshere is meant to refer to Mg²⁺, Mn²⁺, Ca²⁺, ATP, NAD⁺, NADP⁺,diacylglycerol, phosphatidylserine, eicosinoids, retinoic acid,calciferol, ascorbic acid, neuropeptides, enkephalins, endorphins,4-aminobutyrate (GABA), 5-hydroxytryptamine (5-HT), catecholamines,acetyl CoA, S-adenosylmethionine, hexose sugars, pentose sugars,phospholipids, lipids, glycosyl phosphatidyl inositols (GPIs), and anyother biological cofactor.

The pH of the reaction mixture should be between about 1 and 6,preferably between 2 and 3-4 during the multiple HD cycles that will berun. The reaction should be run under anaerobic conditions. Anaerobicconditions may include substantial absence of oxygen including freeoxygen or bound oxygen (NO₂, NO₃), and may include, for convenience, thesequestration of a reaction from atmospheric air. By way ofillustration, anaerobic conditions can be those with less than 1 mgdissolved oxygen per liter of reaction mixture.

The present methods may be used to form double stranded polymers, suchas dsRNA, as shown e.g. in FIG. 12. Generally, a polynucleotide will beused as a template when a predetermined sequence is desired. Otherexamples of double stranded RNA may be found, e.g. in WO 2007031322 A1,“Compositions comprising immunostimulatory RNA oligonucleotides andmethods for producing said RNA oligonucleotides.” Referring now to FIG.12, the possible random single-stranded and double-stranded RNAoligomers are a linear sequence, 122; a double-stranded oligomer, 124; apartially double-stranded oligomer 126; a partially double strandedoligomer with a partially central double-stranded portion 128; apartially double stranded oligomer with a 5′ overhang (could also be 3′overhang) 130; intermediate partial double-stranded structure 132;multiply fragmented double-stranded structure 134; and a hairpinstructure 136.

The polymerization reaction described here can be carried out with about1-20, 1-15 or 1-10, and usually about 1-7 or 4-7 cycles of wetting anddrying. The cycle starts with admixing of the salt solution and nucleicacid materials (monomers and template(s)) and any other excipients toproduce the fluid reaction mixture. The pH of the fluid reaction mixturecan vary, but is usually in a pH range of around 2 to 4. This includes apH of the reaction mixture with fractional increments of 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 over a pH range of about 2, 3, 4,5, and 6. The mixture may be buffered or un-buffered and include one ormore additional excipients such as a detergent and the like.

Drying also may include subjecting the reaction mixture to anon-streaming gas or vacuum (e.g., lyophilization). In anotherembodiment, drying is accomplished by a combination of a stream of gasand lyophilization. Drying refers to the lack of liquid such that thedried material presents a solid appearance. Drying may also be carriedout under variable temperature and/or pH. For instance, the fluidreaction mixture can be dried at a temperature that minimizes inhibitionof polymerization or degradation of the reactants and polymerizationproduct, while maximizing the drying process. Temperature ranges fordrying include below 0 degree C. to around 100° C. Temperature ranges ofspecific interest for drying generally include increments of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 degrees over a range of about 0, 10, 20, 30, 40,50, 60, 70, 80, 90 and 100° C.

While the dried reaction mixture can be immediately rehydrated, thedried reaction mixture may also be allowed to incubate for a period oftime sufficient for polymerization of monomer. Reaction times aregenerally chosen so as to optimize polymerization. Exemplary incubationtimes for the dried reaction mixture are 5 minutes or longer andtypically include increments of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 minutes of a range of about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170or 180 minutes or longer (e.g., overnight, or even longer such as whenplaced in short or long term storage), and more typically 30 minutes to120 minutes. The dried reaction mixture can be sampled and tested,stored for later use, or followed by a rehydration step during which thelipid matrix is re-solvated.

Rehydration of the dried reaction mixture generates a nucleic acidproduct that has been synthesized via a condensation reaction from themononucleotides present in the mononucleotide composition. Rehydrationgenerally takes between 5 and 30 minutes depending on reaction volume,gas exposure and heating. For example, rehydration takes on averageabout 1 minute for a 0.5 ml reaction volume under a stream of carbondioxide at around 90° C. The rehydration solvent can be the same solventsystem employed in the first cycle, and is usually a weak aqueous proticacid solution with or without buffer.

Anaerobic Heating and Drying Cycles

A chamber was constructed to carry out HD cycles under anaerobicconditions (FIG. 2). Glass vials (1.5 mL) containing reaction mixturesto be tested are placed in 24 wells in an aluminum disk, and the chamberis filled with an inert gas such as carbon dioxide. The disk is heatedto a desired temperature, and rotation of the disk is controlled by aprogrammed stepper motor. As the disk rotates in steps of 15 degreesevery 30 minutes, the samples are dehydrated by a flow of dry carbondioxide through four ports on either side of the disk. Each vial isdried for 2 hours as it rotates under the gas flow, then the rotationbrings the vial under a ports through which water flows at a rate of 0.4mL in 15 minutes. The water is injected at a constant rate by aprogrammable syringe pump. The result is that in a 24 hour period everysample undergoes four cycles of wetting and drying. The CO₂ maintainsanaerobic conditions and carries away water molecules produced duringester bond synthesis, thereby preventing back reactions of hydrolysis.

Smaller scale experiments were also carried out using glass slides withtwo wells on each slide that hold 0.1 mL of the reaction mixture. Fourslides could be arranged on a laboratory hot plate set at the desiredtemperature range, and a plastic flow box with 8 small holes (1 mmdiameter) was set on the slides. Each hole was placed directly over awell so that carbon dioxide gas flowed onto the mixture. A flow metermonitored the gas flow which was set at 2 cc/sec into each well. Thepurpose of the gas was to exclude oxygen and to carry away water vaporas it left the reaction mixture. Each HD cycle was 30 minutes, ratherthan the 2 hour cycle in the larger chamber.

The examples below confirm that drying—rehydration cycles at moderatelyelevated temperature ranges provide sufficient chemical potential todrive the synthesis of phosphodiester bonds between nucleosidemonophosphates.

A polymer that has physical and chemical properties of RNA issynthesized by HD cycles, and monovalent salts unexpectedly improveyields by ten-fold or more. Furthermore, it is shown here that RNase Aacts on the presently prepared products of AMP and UMP, with 90%disappearing from gels after a one hour incubation. This resultconfirmed that UMP had been incorporated into the polymer, because RNaseA attacks pyrimidine bonds.

Remarkably, when both AMP and UMP are present, the products appear tohave significant duplex character as indicated by multiple analyticalmethods. This represents the first time that a double stranded nucleicacid has been synthesized in the absence of enzymes or activatedsubstrates. The implication is that such reactions can give rise todouble stranded polymers by what is essentially self-assembly.

In summary, double stranded nucleic acids having random sequences ofnucleotide bases are synthesized by multiple hydration-dehydrationcycles when monovalent salts are present in the mixture. The monovalentsalts include NaCl, KCl, and NH₄Cl. LiCl does not promotepolymerization. The polymers result from a reaction mechanism involvingan acid-catalyzed ester bond synthesis with a pH optimum near 3. Feedingadditional mononucleotides markedly increases yields, supporting theclaim that amplification occurs.

The preparation of libraries of random RNA sequences has a number ofuses. For example, Stich et al., “On the structural repertoire of poolsof short, random RNA sequences,” J. Theoretical Biol. 252(4): 750-763(2008) investigated computationally the structural properties of a largepool (10⁸ molecules) of single-stranded, 35 nt-long, random RNAsequences. They reported that the distribution of RNA structural motifswithin pools of random sequences is extremely heterogeneous, astheoretical studies and observation of natural secondary structuresdemonstrate. A main concern of experimentalists seeking new ribozyme oraptamer activities is how to deviate the structural composition of theinitial pools in the in vitro experiments from average expectations,thus enhancing for instance the presence of rare structures, or forcingthe ensemble to be structurally biased towards specific commonstructures. A library of RNA molecules of length 35 nt consisting ofrandom linear sequences composed of the four types of nucleotides A, C,G, and U as studies may be prepared in an actual pool, instead of merelycomputed as reported by these authors. The pool may then be testedagainst various targets, or otherwise analyzed. It is known that thelength of ligand binding aptamer motifs can be easily reduced to 25-30nt and in some cases to even smaller molecules with as few as 12-13 nt.These lengths are readily achievable using the present methods.

FIG. 12 illustrates the kinds of single stranded and duplex (fully andpartially double-stranded) products that might be present in themixture. These can be identified by using nanopore analysis, massspectra, sequencing, etc. Nanopore analysis will indicate the length andstrandedness of the product. See, e.g. US 20120094278 for details onnanopore polynucleotide analysis.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes ofclarity, the following terms are defined below.

The abbreviation “HD” refers to heating and drying cycles, where thepresent liquid solutions are heated and dried while containing thereactants used to form the nucleic acid polymers. The sample is heatedand dried, then reconstituted, in a period of time that is desirablyabout 0.1 to 6 hours, but may be between 0.1 hours and 20 hours. Dryingmay be carried out by introducing a drying gas to the solution. Heatingmay be to any temperature above room temperature, up to about 100degrees C., but preferably not above 90 degrees C. The present processtypically employs multiple HD steps in a single synthesis.

Ranges: For conciseness, any range set forth is intended to include anysub-range within the stated range, unless otherwise stated. As anon-limiting example, a range of 120 to 250 is intended to include arange of 120-121, 120-130, 200-225, 121-250 etc. The term “about” hasits ordinary meaning of approximately and may be determined in contextby experimental variability. In case of doubt, “about” means plus orminus 5% of a stated numerical value.

The term “solution” as used herein refers to ordinarily solid materialcontained in a liquid carrier. The term is not, unless otherwise noted,limited to true solutions, and may also refer to suspensions or otherliquid mixtures.

The term “mononucleotide” as used herein refers to a single nucleotidethat can be covalently linked to one or more other such entities to forma polymer. In certain embodiments, the mononucleotides have first andsecond sites (e.g., 5′ and 3′ sites) suitable for binding to other likemonomers by means of standard chemical reactions (e.g., nucleophilicsubstitution), and a diverse element which distinguishes a particularmonomer from a different monomer of the same type (e.g., a nucleotidebase, etc.). In the art synthesis of nucleic acids of this type utilizesan initial substrate-bound monomer that is generally used as abuilding-block in a multi-step synthesis procedure to form a completenucleic acid. Exemplary mononucleotides are 5′ adenosine monophosphate(AMP) and 5′ uridine monophosphate (UMP) as monomers, which can bepurchased from Sigma-Aldrich as free acids.

The term “nucleoside” is used in its conventional sense to refer toglycosylamines that can be thought of as nucleotides without a phosphategroup. A nucleotide is composed of a nucleobase (also termed anitrogenous base), a five-carbon sugar (either ribose or deoxyribose),and one or more phosphate groups while a nucleoside consists simply of anucleobase and a 5-carbon sugar. In a nucleoside, the base is bound toeither ribose or deoxyribose via a beta-glycosidic linkage. Examples ofnucleosides include cytidine, uridine, adenosine, guanosine, thymidineand inosine.

The term “nucleotide” is used in its conventional sense as a compoundcomposed of a phosphate group, the bases adenine, cytosine, guanine, andthymine, and a pentose sugar, in RNA the thymine base being replaced byuracil. The term also includes heterocyclic bases that have beenmodified. Such modifications include methylated purines or pyrimidines,acylated purines or pyrimidines, alkylated riboses or otherheterocycles.

In addition, the terms “nucleoside” and “nucleotide” include thosemoieties that contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, or are functionalized as ethers, amines, or the like. The termnucleotide, as is commonly understood, refers to monophosphate,diphosphate, or triphosphate nucleosides.

The term “polymer” means any compound that is made up of two or moremonomeric units covalently bonded to each other, where the monomericunits may be the same or different, such that the polymer may be ahomopolymer or a heteropolymer. Representative polymers includepeptides, polysaccharides, nucleic acids and the like, where thepolymers may be naturally occurring or synthetic.

The term “oligomer” is used herein to indicate a chemical entity thatcontains a plurality of monomers. As used herein, the terms “oligomer”and “polymer” are used interchangeably, as it is generally, although notnecessarily, smaller “polymers” that are prepared using thefunctionalized substrates of the invention, particularly in conjunctionwith combinatorial chemistry techniques. Examples of oligomers andpolymers include polydeoxyribonucleotides (DNA), polyribonucleotides(RNA), other polynucleotides which are C-glycosides of a purine orpyrimidine base, polypeptides (proteins), polysaccharides (starches, orpolysugars), and other chemical entities that contain repeating units oflike chemical structure. In the practice of the instant invention,oligomers will generally comprise about 2-50 monomers, such as about2-20, and including about 3-10 monomers.

The term “nucleic acid” as used herein means a polymer composed ofnucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compoundsproduced synthetically (e.g., PNA as described in U.S. Pat. No.5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides. Unless specified otherwise, the term refersto naturally occurring RNA, as well as modified RNA such as RNAscontaining non-natural nucleosides or sugars. A listing of RNAmodifications may be found, e.g. in “The RNA Modification Database,”http (colon slash slash) mods.rna.albany.edu/home.

The term “oligonucleotide” as used herein denotes single-strandednucleotide multimers of from about 10 up to about 200 nucleotides inlength, e.g., from about 25 to about 300 nucleotides (“nt”), includingfrom about 50 to about 175 nt, e.g. 150 nt in length

The term “polynucleotide” or “polynucleic acid” is used in theconventional sense and refers to single- or double-stranded polymerscomposed of nucleotide monomers, including oligonucleotides, whereinnucleotide monomers are covalently bonded in a chain. DNA(deoxyribonucleic acid) and RNA (ribonucleic acid) are examples ofpolynucleotides with distinct biological function. Although DNA and RNAdo not generally occur in the same polynucleotide, the four species ofnucleotides may occur in any order in the chain.

The term “salt” is used in the conventional sense and refers tomaterials in solid form or in solution formed from an anion(s) and acation(s). The term monovalent refers to an atom, ion, or chemical groupwith a valence of one, which thus can form one covalent bond. Thepresent monovalent salts may include for example, alkali metal salts(e.g. alakali metal halides) double salts, and may include metal salts,such as NaF, CsCl, NaBr, NaClO₄, imidazole hydrochloride, etc., as wellas those listed below.

As used herein a “high salt concentration” can be any concentration ofsalt that is effective to increase polymerization in the absence of anorganizing macromolecule such as a lipid. Preferably, the aqueous highsalt concentration solution can be any concentration from 50 mM to 3M,preferably from about 0.1 M to about 2M. The salt that can be used inthis invention can be sodium chloride, potassium chloride, calciumchloride, and the like. This dilute peracetic acid sterilizing solutionwith high salt concentration does not need to be pH adjusted.

The term “acidic” is used in the conventional sense, i.e. a pH below 7.As exemplified below, and acidic pH range can cover a variety of ranges,e.g. 1-5, 1-6, 2-6, 3-5, 2-5, etc. (see reference to “Ranges” above)

As used herein, the phrase “anerobic” means reaction conditions whereinthe reaction mixture is not exposed to air or oxygen. In variouscontexts, a reaction in anaerobic conditions may refer to a reactionenvironment completely free of oxygen, essentially free of air, freefrom introduction of air, replacement of air by a non-reactive gas, etc.

The term “short interfering RNA” or siRNA is used in the conventionalsense double-stranded RNA that resemble the products produced by DICERand specifically inhibit gene expression in many different mammaliancell lines. Small interfering RNA (siRNA) is typically anoligonucleotide of about 21 nucleotides (also 21 bases) in length whichis used in RNA interference. The process begins with dsRNA (doublestranded RNA) which is broken down with the help of Dicer into smallfragments approximately 21 nt in length. These siRNA fragments have 2nucleotide overhangs on their 3′ ends. Argonaute2 then helps toincorporate siRNA into RISC (RNA-induced silencing complex). This RISCthen binds to and cleaves mRNA, knocking out the corresponding gene.

EXAMPLES Example 1: Reactions in Small HD Apparatus

Two monomers were chosen as a model system—5′-adenosine monophosphateand 5′-uridine monophosphate—in their acid forms rather than as sodiumsalts. When dissolved in water at 10 mM concentration the pH of thesolution is ˜3. These two mononucleotides ordinarily formhydrogen-bonded base pairs in RNA. Polyadenylic acid (polyA) andpolyuridylic acid (polyU) served as polynucleotide standards. These weremixed in 1:1 mole ratios with respect to the bases to produce doublestranded RNA (polyAU).

Reaction Mixtures

A typical reaction mixture in the larger simulation chamber had 0.2 mLof 10 mM mononucleotides and 0.1 M monovalent salts (LiCl, NaCl, KCl,NH₄Cl). Variables that were tested included the initial pH, temperature,and ionic composition. Volumes were reduced to 0.1 mL when glass slideswere used.

Isolation of Products

The polymer products were isolated in two ways: standard precipitationin ethanol, and purification with Invitrogen RNeasy spin tubes. Similaramounts were obtained, consistent with the presence of polymers thatbehaved like RNA. Depending on the conditions, typical yields rangedfrom 1% to 10% expressed as the fraction of the total weight ofmononucleotide present.

Example 2: Analysis of Products

Products of the reaction were initially monitored by gel electrophoresisusing Invitrogen 4% agarose gels with ethidium bromide staining. Someexperiments used hand-poured 2% gels and the same intercalating dye. Allof the gels are shown as inverted images to increase contrast. Productswere also monitored by nanopore analysis, which has single moleculesensitivity. The nanopore method is described in Vercoutere et al. 2001,and De Guzman et al. 2006. Total yields were estimated by NanoDropspectrophotometry. When the conditions were optimized for maximumyields, samples were analyzed by atomic force microscopy, highperformance liquid chromatography (HPLC) and mass spectrometry.

The experiments described below were guided by predictions arising fromthe hypothesis that hydrothermal cycles can drive polymerizationreactions and synthesis of double stranded products:

1. A standard dsRNA must be sufficiently stable to withstand multiple HDcycles at 85 C and pH 3.

2. If dsRNA is among the products, it should bind intercalating dyeslike ethidium bromide during gel electrophoresis.

3. The polymers will exhibit hyperchromicity if duplex species areproducts.

4. Nanopore signals will resemble those expected for duplex species,rather than single stranded oligomers.

5. Atomic force microscopy should reveal short oligomers in the sizerange expected from gel analysis of products.

6. Electrospray and MALDI mass spectrometry of a known dsRNA shouldclosely resemble that of the product.

7. If a template strand is present, the products should containsequences complementary to the template sequences.

8. Amplification of template strands should be observable.

Stability of RNA

It was essential to establish the rate at which a known RNA sampleundergoes hydrolysis during HD cycles at acid pH ranges and elevatedtemperatures. If RNA hydrolyzed completely in a single 2 hour cycle,there would be no reason to look for synthesis. It is generallyconsidered that RNA is a fragile molecule, and most workers in the fieldwould be surprised if extensive hydrolysis of the polyAU duplex standarddid not occur. However, FIG. 4A shows a gel and NanoDrop™ UV-Visanalysis of polyAU duplex going through multiple HD cycles. Most of thepolyAU survived four cycles. The gels and scans (4B) are duplicatesamples exposed to four HD cycles. The experiment began with 20micrograms of polyAU, and the Y axis shows the micrograms of polymerremaining after each cycle (4C). This shows that the hydrolysis of RNApolymers is not occurring during the HD cycles at a rate that willprevent polymerization y formation of the phosphate linkages.

Cycling Increases Yield of Polymers

FIG. 5 shows how the yield increases with the number of cycles,particularly the longer products in the 300 mer range. Also note that asingle cycle does not produce the same yield even though the total time(4 hours) is equivalent to 8 cycles of 30 minute duration.

Monovalent Ions Promote Yields of Polymers

As noted earlier, if the HD cycles are run with monovalent saltspresent, yields of polymer are dramatically increased (FIG. 6). Thesalts are initially at 0.1 M concentration, and sodium, potassium andammonium chloride all seem to catalyze polymerization, with NH₄Cl havingthe greatest effect. Significantly, LiCl is ineffective. The lithiumcation is strongly hydrated, which may explain why it is unable tocatalyze a condensation reaction. The salt effects are summarized inTable 1.

FIG. 6 shows that polymer yields increased in the presence of monovalentsalts. Ammonium chloride was most effective in promoting polymerization,while lithium chloride had virtually no effect. The hours are cumulativetimes of 30 minute HD cycles, so 4 hrs represent 8 cycles.

TABLE 1 Summary of salt effects on polymerization Salt PolymerizationNH₄ H₂PO₃ + NH₄CO₃ + KCl + NaCl ++ NH₄Cl ++++ LiCl (no effect) NH₄molybdate (no effect)

Intercalation of Ethidium Bromide into dsRNA

It is well known that the fluorescence of certain dyes is markedlyenhanced when they intercalate into double stranded polynucleotides.This effect is illustrated in FIG. 7, which shows a gel with polyA,polyU and the duplex species polyAU produced by mixing the twohomopolymers in a 1:1 mole ratio of A:U. The homopolymers (2 μg) do notbind the dye, but simply mixing the polyA and polyU to produce the sameamount of the duplex species polyAU gives a strongly fluorescent banddue to ethidium intercalation. This result is significant, because theproducts of the HD polymerization reaction bind ethidium and arestrongly fluorescent in the gels shown here.

Example 3: Acidic Conditions

An acidic pH is required to synthesize longer strands of polymer in therange of 100-300 nt, as shown in FIG. 7. FIG. 7 shows that a pH in therange of 3 produces more high MW product than processes carried out athigher pH. This is consistent with a mechanism involving acid catalyzedester bond formation (FIG. 8). Although synthesis of the shorteroligomers (20-40 mers) can occur over a broader pH range, there isvirtually no product in alkaline conditions.

Example 4: Confirmation of RNA Polymerization-(RNase Digestion)

The polymers can be digested by RNase A, an enzyme that hydrolyzes thephosphodiester bonds formed by UMP (FIG. 9). This important resultindicates that UMP has become incorporated in an RNA-like polymer thatis recognized by an enzyme specific for RNA.

Example 5: Product Analysis

Hyperchromicity

UV spectra of samples were obtained while heating from room temperatureto 90 degrees C. Most samples showed hyperchromicity that increased withtemperature, then decreased upon cooling. This is consistent with thepresence of double stranded products.

Nanopore Analysis of Products

The majority of blockades seen in a nanopore instrument were in therange of several milliseconds duration and approximately 25% amplitude(data not shown). This duration and amplitude is consistent with thepresence of dsRNA, because single strand duration would be measured intens of microseconds with blockade amplitudes ranging from 80-90%.

Atomic Force Microscopy

A solution of products was dried on freshly cleaved mica sheet, thenrinsed in deionized water and dried again. Images of samples showed whatwould be expected of duplex species in the size range of 50-100 nt,which was shown (FIG. 10).

Products were also analyzed by mass spectrometry. A DNAPac PA 100 columndesigned to separate oligonucleotides was used in conjunction with theelectrospray mass spectrometer facility at UC Santa Cruz. Mass spectraof the standard polyAU duplex and the putative dsRNA productdemonstrated a polymeric structure of the synthesized polynucleic acids(data not shown). The patterns are similar, consistent with the presenceof dsRNA among the products.

Example 6: “Feeding” Experiment (Template Directed Synthesis)

It is possible that dsRNA species were not only synthesized, but in factcould act as templates during multiple cycles, analogous to theamplification of dsDNA in the polymerase chain reaction. To test thisidea, we performed a feeding experiment in which fresh substrates(nucleic acid monomers) were added after 8 or 12 HD cycles of 30minutes, followed by another 8 or 12 cycles. If no templating occurred,the amount of products would double after feeding, but if the existingpolymer strands were serving as templates more than a simple doublingwould be observed. In other words, initial templates form newpolynucleotides that also serve as templates for additionalmononucleotides, creating an exponential increase in polynucleotides.

FIG. 11A shows a gel and a densitometer scan of higher RNA polymersobtained as a result of the feeding of additional AMP and UMP during theprocess. The two lanes on the left are 8 cycles and the two lanes on theright at 12 cycles before and after adding AMP and UMP. A scan of thegel in the lower panel clearly indicates that more than twice the amountof long chain product has been synthesized.

Template directed synthesis can be carried out by adding a desiredtemplate and the required mononucleotides. Using the present descriptionas a guide, one may use a DNA or RNA strand with the desired sequence,put it through multiple HD cycles with mononucleotides present, thenisolate and purify the products. This should work very well tosynthesize siRNA, which is a dsRNA with 20 or so base pairs. Furtherdetails on sequences and uses of such molecules may be found, e.g., inChristian et al. “Short interfering nucleic acid hybrids and methodsthereof,” US 20040053289, published Mar. 18, 2004.

FIG. 12 illustrates the different possible structures of random RNApolymers produced by the present process.

Addition of a template of a desired sequence is carried out by providinga warm temperature, (e.g. 70-90 deg. C.) and the salts, acid, andmonomers described above. In addition a template polynucleotide, that isDNA or RNA is added. For example, equal amounts of dTMP, dGMP, dCMP anddAMP at a total nucleotide concentration of 10 mg/ml are mixed in 0.5 mltotal of MilliQ H₂O+salt. Template oligomer (5 mg) is added and themixture is incubated at 90 deg. C. for 2 h under a continuous gentlestream of CO₂ gas. The CO₂ served to remove water. After rehydrating thereaction mixture for 10 min with 0.5 ml of salt solution, the incubationis repeated. The incubation and rehydration cycle is repeated 5 timesand 0.5 ml MilliQ H₂O was used for the last rehydration. The synthesizedDNA may be purified from the solution and further amplified by PCR.

Example 7: Increasing Polymer Yield Synthesis-Based on Manuscript

As previously, two monomers were chosen adenosine 5′-monophosphate (AMP)and uridine 5′-monophosphate (UMP) in their acid forms rather than assodium salts (Sigma-Aldrich). When dissolved in water at 10 mMconcentration the pH of the solution is ˜2.5. Commercial polyadenylicacid (polyA) and polyuridylic acid (polyU) were used as polynucleotidecontrol standards (Sigma-Aldrich). These were mixed in 1:1 mole ratioswith respect to the bases to produce double stranded RNA (polyA-polyU).The effects on oligomerization of a variety of monovalent salts,including LiCl, NaCl, KCl, and NH₄Cl were tested. During evaporation,the salts formed crystalline films when their solubility was exceeded.The growing crystals excluded other solutes such as the mononucleotides,producing highly concentrated eutectic phases within the salt matrix.

A Laboratory Simulation of HD Cycles

Simulations were carried out using glass slides with two wells on eachslide that hold 0.1 mL of the reaction mixture. Four slides werearranged on a laboratory hot plate set at the desired temperature range,and a plastic flow box with 8 small holes (1 mm diameter) was set on theslides. Each hole was placed directly over a well so that carbon dioxidegas flowed onto the mixture at approximately 1 cc/sec into each well.The gas was used to exclude oxygen, but also to carry away water vaporfrom condensation reaction as ester bonds formed, thereby preventinghydrolytic back reactions.

Reaction Mixtures

Mononucleotides, AMP (10 mM) and UMP (10 mM), were initially mixed in a1:1 volume ratio. The mononucleotides solution and 0.1 M monovalentsalts were mixed in a 2:1 volume ratio so that the initialconcentrations were 3.3 mM AMP and UMP, together with 0.033 M salt.Because water evaporated during dehydration, these dilute solutionsbecome highly concentrated and finally dry, so it is the ratios that aresignificant rather than the initial concentrations. In a typicalexperiment, the reactants were exposed to 1-16 cycles of wetting anddrying. The temperature (85° C.) and flow of carbon dioxide causeddrying within 1-2 minutes. After each dehydration phase of 30 minutes,the samples were dispersed in 0.1 mL of 1.0 mM HCl to maintain acidity,followed by the next dehydration cycle. Variable experimental parametersincluded initial pH, temperature, the time given to each cycle and thenumbers of cycles. At the end of the cycle series, the samples weredissolved in 0.1 mL of water.

Isolation of Products

The polymer products were isolated by standard precipitation in ethanol(2.5×volume ethanol 100%, 1/10 volume sodium acetate 3 M pH 5.2, 1.6 μLlinear acrylamide 5 mg/mL (Fischer scientific) for 700 μL of reactionmixtures, followed by incubation at −20° C. overnight). The pellets wereconsistent with polymers that behaved like RNA. Quantitative analysiswas performed by UV absorbance with a NanoVue instrument calibrated forRNA to estimate yields of products. Depending on the conditions, typicalyields ranged from 1% to 40% expressed as the fraction of the totalweight of mononucleotides present, and over 55% if additional monomerswere added during cycling.

Characterization of Products

As described above, double-stranded polynucleotide structure was shownby ethidium bromide, alkaline hydrolysis, RNase hydrolysation,hypochromicity, nanopore analysis and microscopy.

Effect of Monovalent Cations on Polymerization

When the HD cycles were run with monovalent salts in the reactionmixture, yields of polymer were dramatically increased compared toabsence of salts. Furthermore, the products were stained by ethidiumbromide, an intercalating dye, suggesting that base stacking waspresent. Sodium, potassium and ammonium chloride all promoted synthesisof polymers containing AMP and UMP as monomers. Products ranging from 10to 300 nucleotides with a peak around 100 mers were detected. NH₄Cl hadthe greatest effect, but products from LiCl produced only a weak band inthe gel even though the yield measured by ethanol precipitation was inthe same range as NH₄Cl (Table 2). The A₂₆₀/A₂₈₀ ratio provides anestimate of how much of the absorbance is due to polymers and how muchto monomers. A ratio of 2 corresponds to RNA while a ratio of 3.4 isobserved for monomers. The high ratio with LiCl indicates that theproduct has relatively short strands of oligomer lacking base stackingcompared with the other salts.

Mixtures of AMP 10 mM+UMP 10 mM+monovalent salt 0.1 M (LiCl, KCl, NaCland NH4Cl) in 1:1:1 volume ratio were submitted to 16 HD cycles of 30minutes. Table 2 below shows yields of polymers synthesized and ratioA260/A280 measured by UV absorbance with a NanoVue instrument. Yieldsare values from duplicate samples, and were calculated as the percent byweight of the original AMP and UMP present in the mixture

TABLE 2 Effect of monovalent salts on polymerization. Salt Yield (%)A₂₆₀/A₂₈₀ LiCl 38; 42 3.4 NaCl 16; 18 2.1 KCl 25; 29 2.0 NH₄Cl 34; 372.0

Yields were highest with LiCl, NH₄Cl, KCl and NaCl, in that order, butthe LiCl product was less stained by ethidium, probably because theoligomers were shorter with decreased base stacking.

Cycling Increases Yield of Polymers

The optimum conditions for the polymerization process were determined byperforming a set of experiments using a variety of controls andconditions including the number of cycles, duration of the cycles, pHand temperature. The synthesis of polymers is the most efficient at hightemperature (around 85° C.), at acidic pH (3) and in the presence of CO₂stream. Without wishing to be bound by any theory, the above suggeststhat synthesis of the ester bond is an acid catalyzed mechanism and thatCO₂ plays an essential role in the polymerization process. Most of theproduct appeared to be polymers from 10 to 300 nucleotides long.Finally, the dehydration phase appeared to be essential for thepolymerization process since a minimum of 30 minutes of drying at eachcycle is necessary to synthesize the 300 nt species (data not shown).

Multiple HD cycles, 30 minutes each, were found to be more effectivethan a long single cycle. It is significant that longer productsaccumulate in later cycles, suggesting that ligation of shorter chainsmay be occurring.

Role of NH₄ ⁺ Cations in Promoting Polymerization

Because NH₄Cl seemed to have the greatest effect on yields of polymers,a series of further experiments were conducted. FIG. 3 shows resultswith different ammonium salts, including ammonium phosphate, ammoniummolybdate, and ammonium formate. Only the ammonium formate yieldedpolymers ranging from 10 to 300 nucleotides in length but in lesseramounts compared to ammonium chloride. The importance of the chemicaleffect of the ammonium cation in this polymerization process was alsotested by substituting tetramethylammonium chloride for ammoniumchloride. FIG. 14 shows polymer synthesis after 8 hours of 30 minutes HDcycles. Yields are normalized for comparison, taking the products in thepresence of NH₄C as 1.0. Salts, lane A: NH₄Cl.; lane B: C₁₉H₄₂BrN; laneC: HCO₂NH₄; lane D: (NH₄)6Mo₇O₂₄4H₂O; lane E: NH₄H₂PO₄. FIG. 14 showsthat tetramethylammonium chloride also produced polymers ranging from 10to 300 nucleotides in length but with lower efficiency than ammoniumchloride.

This suggests that NH₄ ⁺ might have chemical effects induced by itsprotons coupled to the ordering effects on the mononucleotides.

Kinetics of Oligomerization

The oligomerization process in the presence of ammonium chloride followsan exponential curve, and reaches a plateau after 30 hours of wet-drycycles with a yield of 40% (FIG. 15). FIG. 15 shows results from Mixtureof AMP 10 mM+UMP 10 mM+NH₄Cl 0.1 M (1:1:1 volume ratio) showing thetotal amount and yield of products over multiple cycles. Each hour hastwo 30 minutes cycles, so 40 hours represents 80 cycles.

Control of Nucleotide Concentration: Nucleotide Feeding

To determine whether the plateau was due to exhaustion of monomers, afeeding experiment was performed in which fresh monomers were addedevery 2 hours (4 cycles). An enhancement of oligomerization occurredwhen cycling is accompanied by regular additions of monomers. A yield of58% is obtained after 5 feeding steps (final concentration ofnucleotides equal to 60 mM) whereas for the same concentration (60 mM)present at the beginning of the experiment, the yield is 37%. This meansthat controlling nucleotides concentration by stepwise additionsenhances the polymerization process in comparison to nucleotide pool atan equivalent concentration.

The plateau can be due to an equilibrium between synthesis andhydrolysis, although degradation of nucleotides over time may alsocontribute. FIG. 6 shows that longer products accumulate in latercycles. Indeed, there is an enhancement of the production of shortfragments (10 and 150 nts) after few cycles, and then their presencedecreases as a function of time whereas the long polymers (700 and 1000nts) accumulate in the later cycles. Lengthening may occur either byelongation and/or by ligation of short fragments.

Example 8: Preparation of a Short Interfering RNA (siRNA)

Hydration/Dehydration Cycles

Processing of reactants was carried out in two ways. The first was inthe robotic cycling apparatus described above. A simpler alternative forsmaller numbers of samples was carried out using glass slides with twowells on each slide that hold 0.1 mL of the reaction mixture. Fourslides were arranged on a laboratory hot plate set at the desiredtemperature range, and a plastic flow box with 8 small holes (1 mmdiameter) was set on the slides. Each hole was placed directly over awell so that carbon dioxide gas flowed onto the mixture at approximately1 cc/sec into each well. The gas was used to exclude oxygen, but also tocarry away water vapor from condensation reaction as ester bonds formed,thereby preventing hydrolytic back reactions.

Reaction Mixtures

Mononucleotides, AMP (10 mM) and UMP (10 mM), were either used singly oras a mixture (1:1 volume ratio). The mononucleotide solution and 0.1 Mmonovalent salts were mixed in a 2:1 volume ratio so that the initialconcentrations were 3.3 mM AMP and UMP, together with 0.033 M salt.Because water evaporated during dehydration, these dilute solutionsbecome highly concentrated and finally dry, so it is the ratios that aresignificant rather than the initial concentrations. In a typicalexperiment, the reactants were exposed to 1-16 cycles of wetting anddrying. The temperature (85° C.) and flow of carbon dioxide causeddrying within 1-2 minutes. After each dehydration phase of 30 minutes,the samples were dispersed in 0.1 mL of 1.0 mM HCl to maintain acidity,followed by the next dehydration cycle. At the end of the cycle series,the samples were dissolved in 0.1 mL of water and yields were determinedwith a Nanodrop instrument. Composition was monitored by gelelectrophoresis using ethidium bromide as a marker for double strandedproducts, and the presence of single stranded products was confirmed bynanopore analysis.

Introduction of Templates

When it was desired to introduce templates, the nucleic acid templateare added to the reaction mixture and processed as described above. Inone example, 20 micrograms of polyuridylic acid was added together withdAMP. Approximately 30 micrograms of product was recovered by ethanolprecipitation after 4 cycles. An aliquot of the product was analyzed bygel electrophoresis using ethidium bromide as an intercalating dye, anda long fluorescent streak appeared, indicating that the dye had staineda double stranded product. The streak began at the 20 mer range asindicated by a DNA ladder, and extended nearly to the top of the gel.This would be expected because polyU is a mixture of shorter and longerhomopolymers.

SiRNA

Either a known siRNA can be used, or a siRNA can be designed usingavailable software. See, e.g. US 20140161894, “Sirna silencing of genesexpressed in cancer,” US 20050058982, “Modified small interfering RNAmolecules and methods of use,” U.S. Pat. No. 8,318,689, “SiRNA-basedcancer treatment,” U.S. Pat. No. 7,947,659, “iRNA agents targetingVEGF,” etc.

The template is a polynucleotide that is DNA and encodes both strands ofthe desired siRNA. It may further comprise intervening sequences betweenthe two RNA sequences being formed on the template. The template mayinclude restriction enzyme sited between the two strands being formed,to facilitate assembly of the two complementary strands of the siRNA.The template then will be about 50-100 in length and will besingle-stranded.

The necessary RNA monomers are incubated with a number of templatemolecules in the acidic/heat/salt combination described above.

The following siRNA construct is prepared for study. It inhibitsexpression of the luc gene. Sequence of the luc gene (may be found, forexample, at GenBank: KJ081213.1. The firefly luciferase gene is furtherdescribed at Wetr et al., “Firefly Luciferase Gene: Structure andExpression in Mammalian Cells,” Mol. Cell. Biol., 7(2):725-737 (1987).As described there, luciferase expression is a means of monitoringexpression of a gene. It may be used here to measure the uptake andeffectiveness of an SiRNA product. The siRNA prepared has the followingsequences:

(SEQ ID NO: 1) 5′-ACGCCAAAAACAUAAAGAAAG-3′ (SEQ ID NO: 2)3′-UCUGCGGUUUUUGUAUUUCUU-5′

To produce this above duplex RNA, one may synthesize a singlesingle-stranded DNA template (e.g SEQ ID NO: 3) or a pair of ssDNAtemplates. The templates incorporate sequences for the two complementarybase sequences. e.g. SEQ ID NO: 1 and SEQ ID NO: 2 above. If a singletemplate is used for both RNAs, an abasic nucleotide may be used toseparate the sequences encoding the two siRNA strands. Restriction sitesmay also be engineered into a single template.

Further constructs may be designed as described at https (colon slashslash) www (dot)broadinstitute.org/rnai/public/gene/details?geneld=TRCG0000060314.

The DNA template is cycled multiple times under the above-describedconditions of salt, acidity, heat, atmosphere, etc., with all fourribonucleotide monophosphates present, and each cycle synthesizes thedesired complementary RNA 21 mers that will assemble into duplexstrands. Significantly, the DNA template can be covalently attached tosilica beads so that product RNA can simply be washed off after eachcycle. Errors are likely to occur as the mononucleotides are polymerizedon the template, but the RNA strands with the correct sequence will formstable duplex species that can be easily purified, for instance byelectrophoresis.

Other RNA Products

The present examples also can be applied to other RNA therapeuticmolecules, using the template-driven in vitro synthesis described here.RNA products can be, for example, RNA aptamers, e.g. as described inWang et al., “Aptamers as therapeutics in cardiovascular diseases.,”Curr Med Chem. 2011; 18(27):4169-74. Also contemplated are RNAantisense, e.g. as described in Weiss et al, “Antisense RNA gene therapyfor studying and modulating biological processes. Cell. Mol. Life Sci.,55:334-358, 1999. Also, microRNAs are made by Dicer, but microRNA derivefrom single-stranded RNAs that fold back on themselves to generate smallregions of double-stranded RNA-so called “stem-loops”—instead of thelong double-stranded RNA that produces siRNAs. Most anti-miRs usemodifications of the typical nucleic acid ribose sugar backbone with 2′modifications. Such modified nucleotides can be incorporated using thepresent, non-enzymatic methods. (See, Montgomery et al., “Therapeuticinhibition of mir-208a improves cardiac function and survival duringheart failure,” Circulation. 2011; 124:1537-1547

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification areintended to convey details of methods and materials useful in carryingout certain aspects of the invention which may not be explicitly set outbut which would be understood by workers in the field. Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference andcontained herein, as needed for the purpose of describing and enablingthe method or material referred to.

What is claimed is:
 1. A composition for preparing a polynucleotide frommononucleotides comprising: an acidic solution or a dried solid preparedfrom the acidic solution, free of polymerase and lipids, and comprising:a monovalent salt, wherein the monovalent salt is a metal halide salt oran ammonium halide salt at a concentration of at least 0.01 M; andmononucleotides selected from the group consisting of adenosine5′-monophosphate, uridine 5′-monophosphate, guanosine 5′-monophosphate,and cytidine-5′-monophosphate, or 2′-deoxyadenosine 5′-monophosphate,thymidine 5′-monophosphate, 2′-deoxyguanosine 5′-monophosphate, and2′-deoxycytidine-5′-monophosphate, each at a concentration of from0.001M to 3M.
 2. The composition of claim 1, wherein the mononucleotidesare selected from the group consisting of adenosine 5′-monophosphate,uridine 5′-monophosphate, guanosine 5′-monophosphate, andcytidine-5′-monophosphate, and wherein the polynucleotide is ribonucleicacid (RNA).
 3. The composition of claim 1, wherein the mononucleotidesare selected from the group consisting of 2′-deoxyadenosine5′-monophosphate, thymidine 5′-monophosphate, 2′-deoxyguanosine5′-monophosphate, and 2′-deoxycytidine-5′-monophosphate, and wherein thepolynucleotide is deoxyribonucleic acid (DNA).
 4. The composition ofclaim 1, further comprising a template polynucleotide complementary tothe polynucleotide.
 5. The composition of claim 1, wherein thecomposition comprises an acidic solution.
 6. The composition of claim 5,wherein the acidic solution is adjusted to a pH between 2 and
 4. 7. Thecomposition of claim 5, wherein the monovalent salt concentration isbetween 0.05 and 2M in solution.
 8. The composition of claim 5, whereinthe monovalent salt is a metal halide salt.
 9. The composition of claim8, wherein the metal halide salt is selected from the group consistingof NaF, CsCl, NaBr, NaClO₄, NaCl, LiCl, and KCl.
 10. The composition ofclaim 9, wherein the metal halide salt is present in the acidic solutionat a concentration between 0.05 M and 2M.
 11. The composition of claim5, wherein the monovalent salt is an ammonium halide salt.
 12. Thecomposition of claim 11, wherein the ammonium halide salt is NH₄Cl. 13.The composition of claim 12, wherein the NH₄Cl is present in the acidicsolution at a concentration between 0.05 M and 2M.
 14. The compositionof claim 5, wherein the acidic solution is maintained within atemperature range of 60° C. and 90° C.
 15. The composition of claim 1,wherein the composition comprises a dried solid prepared from the acidicsolution.
 16. The composition of claim 15, wherein the monovalent saltis a metal halide salt.
 17. The composition of claim 15, wherein themetal halide salt is selected from the group consisting of NaF, CsCl,NaBr, NaClO₄, NaCl, LiCl, and KCl.
 18. The composition of claim 15,wherein the monovalent salt is an ammonium halide salt.
 19. Thecomposition of claim 18, wherein the ammonium halide salt is NH₄Cl. 20.The composition of claim 15, wherein the dried solid is maintainedwithin a temperature range of 60° C. and 90° C.